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Team:UESTC-China/Protocol
From 2014.igem.org
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#SectionTitles5{ | #SectionTitles5{ | ||
position:relative; | position:relative; | ||
| + | top: 1500px; | ||
left:30px; | left:30px; | ||
| - | |||
padding:0 10px; | padding:0 10px; | ||
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<td class="mid"> 1ul</td> | <td class="mid"> 1ul</td> | ||
</tr> | </tr> | ||
| + | </table> | ||
| + | </div> | ||
| + | <div id="SectionTitles5" style="width:500px;"> | ||
| + | <table border="1" id="BiosensorsTable" cellspacing="0";> | ||
| + | <tr> | ||
| + | <th class="top"></th> | ||
| + | <th class="top">Temperature </th> | ||
| + | <th class="top">Time </th> | ||
| + | <th class="top"> Cycle</th> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td class="mid">Step1</td> | ||
| + | <td class="mid">94℃</td> | ||
| + | <td class="mid">30s</td> | ||
| + | <td class="mid">X35 cycles</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td class="mid"></td> | ||
| + | <td class="mid"> 56℃</td> | ||
| + | <td class="mid">30s</td> | ||
| + | <td class="mid"></td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td class="mid"></td> | ||
| + | <td class="mid">68℃</td> | ||
| + | <td class="mid">30s/kb</td> | ||
| + | <td class="mid"></td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td class="mid">Step3</td> | ||
| + | <td class="mid">68℃</td> | ||
| + | <td class="mid">5min</td> | ||
| + | <td class="mid">X1 cycles</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td class="mid"></td> | ||
| + | <td class="mid">10℃</td> | ||
| + | <td class="mid">10min</td> | ||
| + | <td class="mid"></td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td class="mid">10uM reverse primer</td> | ||
| + | <td class="mid"> 1ul</td> | ||
| + | <td class="mid">ddH2O</td> | ||
| + | <td class="mid">33ul</td> | ||
| + | </tr> | ||
| + | |||
</table> | </table> | ||
</div> | </div> | ||
Revision as of 05:03, 24 September 2014
DNA ligation with T4 DNA Ligase
| Component | 20ul Reaction |
|---|---|
| 1.33X Master Mix | 15ul |
| Insert DNA | the moles of insert DNA to vector DNA is 5：1 |
| >Vector DNA | 50ng |
| ddH2O | To 20ul |
| Incubate at 50℃ for 1 hour |
DNA ligation with Gibson Assemble
| Component | 20ul Reaction |
|---|---|
| Vector DNA | 50ng |
| Insert DNA | the moles of insert DNA to vector DNA is 5：1 |
| 10X T4 DNA ligase buffer | 2ul |
| T4 DNA ligase | 1ul |
| ddH2O | to 20ul |
| Incubate at 25℃ for 12 hours |
Double Digestion of DNA
| Component | 50ul Reaction |
|---|---|
| 10x FastDigest buffer | 5ul |
| Restriction enzyme 1 | 1ul |
| Restriction enzyme 2 | 1ul |
| DNA | 1-2ug |
| ddH2O | to 50ul |
| Incubate at 37℃ for 2 hours |
PCR protocol for KOD-Plus-Neo
| Component | 50ul Reaction |
|---|---|
| ddH2O | 33ul |
| 10X buffer for KOD-Plus-Neo | 5ul |
| MgSO4 | 1ul |
| 10mM dNTPs | 1ug |
| 10uM forward primer | 1ul |
| 10uM reverse primer | 1ul |
| Template | 1ul |
| KOD enzyme | 1ul |
| Temperature | Time | Cycle | |
|---|---|---|---|
| Step1 | 94℃ | 30s | X35 cycles |
| 56℃ | 30s | ||
| 68℃ | 30s/kb | ||
| Step3 | 68℃ | 5min | X1 cycles |
| 10℃ | 10min | ||
| 10uM reverse primer | 1ul | ddH2O | 33ul |
